Common matrix metalloproteinase 2 gene haplotypes may modulate left ventricular remodelling in hypertensive patients

Matrix metalloproteinases (MMPs) are involved in cardiac remodelling. We examined whether MMP-2 genetic polymorphisms are associated with hypertension and left ventricular (LV) remodelling in hypertensive patients. We studied 160 hypertensive patients and 123 healthy controls. Echocardiography was performed in all patients and the C-1306T (rs243865) and C-735T (rs 2285053) MMP-2 polymorphisms were analysed. Haplo.stats analysis was used to evaluate whether MMP-2 haplotypes are associated with hypertension and with extremes in LV mass index (LVMI). Multiple linear regression analysis was performed to assess whether MMP-2 genotypes or haplotypes affect LVMI and other echocardiography parameters. The C-1306T 'CC' genotype was associated with reduced LVMI and LV end-diastolic diameter (EDD) (P=0.0365 and P=0.0438, respectively). The haplotype 'C, C' was associated with reduced LVMI and EDD (P=0.0278 and P=0.0322, respectively). The comparison of upper and lower extremes of the LVMI phenotype showed that the 'C, C' haplotype was more common in the lower LVMI group (P=0.0060), whereas the 'T, C' haplotype was more common in the higher quartile of LVMI (P=0.0187), and this haplotype was associated with increased risk of higher LVMI values (odds ratio=3.5121, 95% confidence interval 1.3193-9.3494). The findings suggest that MMP-2 polymorphisms affect hypertension-induced LV remodelling. Journal of Human Hypertension (2012) 26, 171-177; doi:10.1038/jhh.2011.8; published online 10 February 2011

Mannose-binding lectin and MBL-associated serine protease-2 gene polymorphisms in a Brazilian population from Rio de Janeiro

Mannose-binding lectin (MBL) is a protein able to bind to carbohydrate patterns on pathogen membranes; upon MBL binding, its associated serine protease MBL-associated serine protease type 2 (MASP2) is autoactivated, promoting the activation of complement via the lectin pathway. For both MBL2 and MASP2 genes, the frequencies of polymorphisms are extremely variable between different ethnicities, and this aspect has to be carefully considered when performing genetic studies. While polymorphisms in the MBL-encoding gene (MBL2) have been associated, depending upon ethnicity, with several diseases in different populations, little is known about the distribution of MASP2 gene polymorphisms in human populations. The aim of our study was thus to determine the frequencies of MBL2 (exon 1 and promoter) and MASP2 (p.D371Y) polymorphisms in a Brazilian population from Rio de Janeiro. A total of 294 blood donor samples were genotyped for 27 polymorphisms in the MBL2 gene by direct sequencing of a region spanning from the promoter polymorphism H/L rs11003125 to the rs1800451 polymorphism (at codon 57 in the first exon of the gene). Genotyping for MASP2 p.D371Y was carried out using fluorogenic probes. To our knowledge, this is the first study reporting the prevalence of the MASP2 p.D371Y polymorphism in a Brazilian population. The C allele frequency 39% is something intermediate between the reported 14% in Europeans and 90% in Sub-Saharan Africans. MBL2 polymorphisms frequencies were quite comparable to those previously reported for admixed Brazilians. Both MBL2 and MASP2 polymorphisms frequencies reported in our study for the admixed Brazilian population are somehow intermediate between those reported in Europeans and Africans, reflecting the ethnic composition of the southern Brazilian population, estimated to derive from an admixture of Caucasian (31%), African (34%) and Native American (33%) populations. In conclusion, our population genetic study describes the frequencies of MBL2 and MASP2 functional SNPs in a population from Rio de Janeiro, with the aim of adding new information concerning the distribution of these SNPs in a previously unanalysed Brazilian population, thus providing a new genetic tool for the evaluation of the association of MBL2 and MASP2 functional SNPs with diseases in Brazil, with particular emphasis on the state of Rio de Janeiro.

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Effects of Matrix Metalloproteinase (MMP)-2 Polymorphisms on Responsiveness to Antihypertensive Therapy of Women with Hypertensive Disorders of Pregnancy

Imbalanced matrix metalloproteinase (MMP) expression, including MMP-2, has been demonstrated in pre-eclampsia. However, little is known about the effect of polymorphisms in MMP-2 gene on hypertensive disorders of pregnancy. We examined whether two functional MMP-2 polymorphisms (g.-1306C>T and g.-735C>T) are associated with pre-eclampsia and/or gestational hypertension and whether these polymorphisms affect therapeutic responses in women with these conditions. We studied 216 healthy pregnant women (HP), 185 patients with gestational hypertension (GH) and 216 patients with pre-eclampsia (PE). They were stratified as responsive or non-responsive to antihypertensive therapy according to clinical and laboratorial parameters of therapeutic responsiveness. Genomic DNA was extracted from whole blood and genotypes for g-1306C>T and g.-735C>T polymorphisms were determined by real-time PCR using Taqman allele discrimination assays. Haplotype frequencies were inferred using the PHASE 2.1 program. The distributions of MMP-2 genotypes and haplotypes were similar in HP, GH and PE patients (p > 0.05). In addition, we found no significant differences in MMP-2 genotype or haplotype frequencies when GH or PE patients were classified as responsive or non-responsive to antihypertensive therapy (p > 0.05). Our results suggest that MMP-2 polymorphisms do not affect the susceptibility to hypertensive disorders of pregnancy. In parallel, MMP-2 polymorphisms apparently do not affect the responsiveness to antihypertensive therapy of women with these hypertensive disorders of pregnancy.

Matrix Metalloproteinase (MMP)-2 Genetic Variants Modify the Circulating MMP-2 Levels in End-Stage Kidney Disease

Background: Matrix metalloproteinases (MMPs) play important roles in the pathophysiology of renal diseases, and imbalanced MMP-2 and its endogenous inhibitor (the tissue inhibitor of metalloproteinases-2; TIMP-2) are implicated in the vascular alterations of end-stage kidney disease (ESKD) patients. We have examined whether MMP-2 gene polymorphisms and haplotypes modify MMP-2 and TIMP-2 levels in ESKD patients as well as the effects of hemodialysis on the concentrations of these biomarkers. Methods: We determined MMP-2 and TIMP-2 plasma levels by gelatin zymography and ELISA, respectively, in 98 ESKD patients and in 38 healthy controls. Genotypes for two relevant MMP-2 polymorphisms (C-T-1306 and C-T-735 in the promoter region) were determined by TaqMan (R) allele discrimination assay and real-time polymerase chain reaction. The software program PHASE 2.1 was used to estimate the haplotype frequencies. Results: We found increased plasma MMP-2 and TIMP-2 levels in ESKD patients compared to controls (p<0.05), and hemodialysis decreased MMP-2 (but not TIMP-2) levels (p<0.05). The T allele for the C-T-735 polymorphism and the C-T haplotype were associated with higher MMP-2 (but not TIMP-2) levels (p<0.05), whereas the C-T-1306 had no effects. Hemodialysis decreased MMP-2 (but not TIMP-2) levels independently of MMP-2 genotypes or haplotypes (p<0.05). Conclusions: MMP-2 genotypes or haplotypes modify MMP-2 levels in ESKD patients, and may help to identify patients with increased MMP-2 activity in plasma. Hemodialysis reduces MMP-2 levels independently of MMP-2 genetic variants. Copyright (C) 2012 S. Karger AG, Basel

DSD Due to 5 alpha-Reductase 2 Deficiency - from Diagnosis to Long Term Outcome

Most of the patients with 5 alpha-RD 2 deficiency are reared in the female social sex due to their severely undervirilized external genitalia but similar to 60% who have not been submitted to orchiectomy in childhood undergo male social sex change at puberty. In our cohort of 30 cases from 18 families, all subjects were registered in the female social sex except for two children-one who had an affected uncle and the other who was diagnosed before being registered. The majority of the patients were satisfied with the long-term results of their treatment and surprisingly, penile length was not associated with satisfactory or unsatisfactory sexual activity. Steroid 5 alpha-RD2 deficiency should be included in the differential diagnosis of all newborns with 46,XY DSD with normal testosterone production before gender assignment or any surgical intervention because these patients should be considered males at birth.

Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306 C> T and g.-735 C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306 C>T and g.-735 C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306 C>T and g.-735 C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined. (C) 2012 Elsevier Inc. All rights reserved.

Infrared (810-nm) low-level laser therapy on rat experimental knee inflammation

Arthritis of the knee is the most common type of joint inflammatory disorder and it is associated with pain and inflammation of the joint capsule. Few studies address the effects of the 810-nm laser in such conditions. Here we investigated the effects of low-level laser therapy (LLLT; infrared, 810-nm) in experimentally induced rat knee inflammation. Thirty male Wistar rats (230-250 g) were anesthetized and injected with carrageenan by an intra-articular route. After 6 and 12 h, all animals were killed by CO(2) inhalation and the articular cavity was washed for cellular and biochemical analysis. Articular tissue was carefully removed for real-time PCR analysis in order to evaluate COX-1 and COX-2 expression. LLLT was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity with 1, 3, and 6 J (Joules) of energy. This result was corroborated by cell counting showing the reduction of polymorphonuclear cells at the inflammatory site. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Both COX-1 and 2 gene expression were significantly enhanced by laser irradiation while PGE(2) production was inhibited. Low-level laser therapy operating at 810 nm markedly reduced inflammatory signs of inflammation but increased COX-1 and 2 gene expression. Further studies are necessary to investigate the possible production of antiinflammatory mediators by COX enzymes induced by laser irradiation in knee inflammation.

Analysis of the concordance rates between core needle biopsy and surgical excision in patients with breast cancer

Objective: To evaluate whether immunohistochemical marker studies performed on core needle biopsy (CNB) specimens accurately reflect the marker status of the tumor obtained from final surgical specimen. Methods: This was a retrospective study that used the database of the Division of Mastology of the Hospital das Clinicas, Sao Paulo, Brazil. Sixty-nine patients submitted to ultrasound-guided CNB diagnosed with breast cancer were retrospectively analyzed. Immunohistochemistry (IHC) on core biopsy specimens was compared to that of excisional biopsy regarding estrogen receptor (ER), progesterone receptor (PR), human epidermal gowth factor receptor 2 gene (HER2), p53, and Ki67. The analysis of the concordance between CNB and surgical biopsy was performed using the kappa (k) coefficient (95% CI). Results: A perfect concordance between the labeling in the surgical specimens and the preoperative biopsies in p53 (k = 1.0; 95% CI: 0.76-1.0) was identified. There was an almost perfect concordance for ER (k = 0.89; 95% CI: 0.65-1.0) and a substantial concordance for PR (k = 0.70; 95% CI: 0.46-0.93). HER2 (k = 0.61; 95% CI: 0.38-0.84) and Ki-67 (k = 0.74; 95% CI: 0.58-0.98) obtained a substantial concordance this analysis. Conclusion: The results of this study indicate that the immunohistochemical analysis of ER, PR, Ki-67, and p53 from core biopsy specimens provided results that accurately reflect the marker status of the tumor. The concordance rate of HER2 was less consistent; although it produced substantial concordance, values were very close to moderate concordance.

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.

Molecular epidemiology of the SH (small hydrophobic) gene of human respiratory syncytial virus (HRSV), over 2 consecutive years

Human respiratory syncytial virus (HRSV) strains were isolated from nasopharyngeal aspirates collected from 965 children between 2004 and 2005, yielding 424 positive samples. We sequenced the small hydrophobic protein (SH) gene of 117 strains and compared them with other viruses identified worldwide. Phylogenetic analysis showed a low genetic variability among the isolates but allowed us to classify the viruses into different genotypes for both groups, HRSVA and HRSVB. It is also shown that the novel BA-like genotype was well segregated from the others, indicating that the mutations are not limited to the G gene. (C) 2011 Elsevier B.V. All rights reserved.

Gene Expression Profile in Response to Doxorubicin-Rapamycin Combined Treatment of HER-2-Overexpressing Human Mammary Epithelial Cell Lines

HER-2-positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2-overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G(2)-M, whereas SKBR3 cells showed an increase in the G(0)-G(1) phase. Rapamycin increased the sensitivity to doxorubicin in HER-2-overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2-overexpressing breast cancers to doxorubicin and rapamycin combination. Mol Cancer Ther; 11(2); 464-74. (C) 2011 AACR.

Prognostication of Soft Tissue Sarcomas Based on Chromosome 17q Gene and Protein Status: Evaluation of TOP2A, HER-2/neu, and Survivin

Topoisomerase 2 alpha (), HER-2/ and are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value. Eighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray. TOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes. These findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Abstract Background In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.